Proposal Summary/Abstract Congenital absence or injury to peripheral and cranial nerves yields devastating consequences. This is especially true of the facial nerve as loss of facial function leads to impaired blink impacting sight, nasal valve collapse impacting respiration, lip flaccidity impacting articulation and oral competence, and impaired facial expression with profound negative impacts on emotional and social well-being. As peripheral nerves are capable of regeneration, surgery may be employed to restore critical sensory and motor function. In patients with facial palsy, smile may be reanimated by interposing a nerve autograft across the upper lip to route axons from a healthy-side facial nerve donor branch to the paralyzed side. In a subsequent surgery, free muscle is transplanted into the paralyzed side and neurotized by the cross-facial nerve graft. Such long nerve grafting procedures carry a 20-30% failure rate, believed to be secondary to length-dependent axonal growth arrest. Emerging evidence suggests nerve regeneration over long distances is hampered by progressive downregulation of pro-regenerative transcription factors within axotomized neurons and Schwann cells residing in the distal portions of long nerve grafts. There is a critical need for novel therapeutic strategies to improve peripheral nerve regeneration over long distances. Our long-term goal is to employ gene therapy techniques to prolong pro-regeneration states of Schwann cells and neurons to enhance axonal penetration across long nerve grafts. Though adeno-associated virus (AAV) vectors have been employed for gene delivery to peripheral nerves, transduction efficiency has heretofore remained suboptimal and the facial nerve has not been specifically studied. Further, delivery to transected peripheral nerves has not been investigated. Recently, use of hybrid gene delivery vectors comprising AAV vectors and endogenous nanoparticles termed exosomes (exo-AAV) has demonstrated impressive improvement in gene delivery to sensory hair cells of the inner ear and neurons of the central nervous system. The goal of the proposed research is to characterize differences in the efficiency of transduction of Schwann cells and axons of transected facial nerve by emerging AAV and exo-AAV vectors. We hypothesize that improved fluorescent reporter transgene transduction will be demonstrated for axotomized facial neurons and Schwann cells transfected with exo-AAV as opposed to AAV vectors. A double homozygous transgenic (dTg) fluorescent reporter mouse expressing cyan fluorescent protein (CFP) at high concentrations in peripheral axons and Venus protein in Schwann cells will be employed for high-throughput stain-free assessment of vector transduction efficiency. Results will inform the potential clinical use of AAV or exo-AAV vectors for targeted gene delivery to injured facial nerves.